Project 3 - Mapping Long-range Allosteric Pathways in CRISPR-Cas9

项目摘要 基因调控机制对于适当的细胞和蛋白质功能是至关重要的，并且现代分子生物学也是如此。 生物学将许多病理与这些过程的失调联系起来。虽然修改 纠正致病突变的基因组是一种有前途的治疗方法，这些努力不能 如果不了解CRISPR等蛋白质机器的基本生物化学， Cas9（Cas9）。Cas9可以是编辑和校正疾病相关（基因组）突变的可定制工具， 然而，为了充分实现这些应用，需要新的策略来克服其脱靶效应和不良反应。 必须研究时间控制。Cas9利用向导RNA分子来募集、稳定和表达RNA。 在识别众所周知的前间区序列邻近基序后促进双链DNA的切割 (PAM)顺序先前的X射线晶体结构表明Cas9内的构象变化 核酸酶HNH和RuvC是有效催化功能所必需的。然而，这些结构提供 几乎没有机制信息，因为在一个细胞中从未观察到靶DNA和催化核酸酶。 激活状态。特别是HNH的构象变化与邻近分子的运动有关。 亚结构域，所有这些都被激活，从>20埃以外的PAM结合结构域，这表明， 变构机制了解这种变构耦合将具有令人兴奋的精确潜力 通过建立新的范式来控制和增强空间和时间功能， Cas9我们最近发现了一条跨越HNH核酸酶的毫秒级运动路径 计算结果表明，到达多个Cas9结构域是一个更大的变构蛋白的一部分。 控制Cas9功能的网络。为了研究这种变构网络的范围以及 分子运动的机制，我的实验室将进行协同解决核磁共振， 计算研究以绘制Cas9的远程变构途径。我们现在将（1）描述 已知改变Cas9特异性的HNH的变构突变体，（2）确立了HNH的生物物理作用， 邻近的REC 2和REC 3结构域在向/从HNH传播变构信号中，以及（3） 表征支配全长Cas9蛋白的构象系综。这种多学科 核磁共振自旋弛豫实验，分子动力学模拟和网络理论的方法，将 探测Cas9中的多时间尺度蛋白质运动，揭示负责传递的特定氨基酸 结构或动态信息。这些研究将使用全长Cas9和新的工程改造的Cas9。 构建体以询问160 kDa酶内的特定结构域。结构和动态 这项工作的结果将与新的体内测定功能相关，以提供详细的 了解Cas9变构机制。

Project Summary Gene regulatory mechanisms are critical for proper cellular and protein function, and modern molecular biology has linked numerous pathologies to dysregulation of these processes. Although modification of the genome to correct pathogenic mutations is a promising therapeutic approach, these efforts cannot be successful without knowledge of the underlying biochemistry of protein machinery such as CRISPR- Cas9 (Cas9). Cas9 can be a customizable tool to edit and correct disease-linked (genomic) mutations, however, to fully realize these applications, novel strategies to overcome its off-target effects and poor temporal control must be investigated. Cas9 utilizes a guide RNA molecule to recruit, stabilize, and facilitate cleavage of double-stranded DNA after recognition of a well-known protospacer adjacent motif (PAM) sequence. Prior X-ray crystal structures indicate that conformational changes within the Cas9 nucleases, HNH and RuvC, are required for effective catalytic function. However, these structures offer little mechanistic information, as the target DNA and catalytic nucleases are never observed in an activated state. The conformational shift of HNH, in particular, is correlated to motions of neighboring subdomains, all of which are activated from >20 Å away by the PAM-binding domain, suggesting an allosteric mechanism. Understanding this allosteric coupling would have exciting potential for precision medicine by establishing novel paradigms to control and enhance the spatial and temporal function of Cas9. We recently identified a pathway of millisecond timescale motions spanning the HNH nuclease and reaching multiple Cas9 domains that computational results suggest is a portion of a larger allosteric network that controls Cas9 function. To investigate the reach of this allosteric network and the role of molecular motions in its mechanism, my laboratory will undertake a synergistic solution NMR and computational study to map the long-range allosteric pathway of Cas9. We will now (1) characterize allosteric mutants of HNH that are known to alter Cas9 specificty, (2) establish the biophysical roles of the neighboring REC2 and REC3 domains in propagating allosteric signals to/from HNH, and (3) characterize the conformational ensemble governing the full-length Cas9 protein. This multidisciplinary approach of NMR spin relaxation experiments, molecular dynamics simulations, and network theory, will probe multi-timescale protein motions in Cas9, revealing specific amino acids responsible for transmitting structural or dynamic information. These studies will use both full-length Cas9 and novel engineered constructs to interrogate specific domains within the 160 kDa enzyme. The structural and dynamic findings of this work will be correlated to function with new in vivo assays to provide a detailed understanding of the Cas9 allosteric mechanism.